人巨细胞病毒UL97基因耐药重组株的构建与鉴定

Construction and identification of recombinant human cytomegaiovirus with ganciclovir-resistance associated mutations in UL97 gene

目的构建含UL97基因耐药相关突变的重组人巨细胞病毒（rHCMV），并通过耐药表型和基因型加以鉴定。方法采用重叠延伸剪接PCR在UL97基因中引入PineI位点和耐药相关位点做定点突变，将所获突变基因片段按比例与人巨细胞病毒（HCMV）AD169标准株DNA混合，经脂质体介导共转染成纤维细胞MRC-5。利用间接免疫荧光检测HCMVPP65抗原，证实MRC-5已被rHCMV感染，观察同源重组病毒形成的细胞病变达100％后收获该重组病毒。用不同浓度更昔洛韦进行噬斑筛选纯化目的病毒，并通过噬斑减少试验和耐药基因（UL97和UL54）序列分析对重组病毒进行鉴定。结果成功构建目的突变UL97基因片段，同病毒基因组DNA共转染7d后可见明显细胞病变，PP65抗原检测证实为rHCMV感染灶。经过克隆筛选得到的重组病毒株UL97基因型分析结果与预期一致，UL54基因测序未发现突变。阳性克隆重组病毒对更昔洛韦敏感性显示半数抑制浓度（IC50）为15μmol／L，是标准株的12倍，具耐药表型。结论成功将HCMV基因组DNA与耐药突变目的基因片段共转染MRC-5细胞，通过更昔洛韦筛选和噬斑纯化获得目的重组病毒株。该方法的建立为在用药过程中不断出现的新的HCMV耐药突变株的鉴定分析提供了有效的技术平台。

Objective To construct drug-resistant variant recombinant human cytomegalovirus （rHCMV） and identify drug susceptibility by phenotypic and genotypic assays. Methods The UL97 fragments containing Prne I recongnition site and resistant mutation were introduced by site-directed mutagenesis using gene splicing by overlap extension polymerase chain reaction （PCR）, and blended with human eytomegalo-virus （HCMV） standard strain AD169 genomic DNA proportionally, then the DNA mixture were transfected into MRC-5 fibroblasts by the vector of liposomes. HCMV-PP65 antigen was detected by indirect-immunofluorescent assay to verify rHCMV infection of MRC-5 fibroblasts. When the cytopathic effect （CPE） of homologous recombinant virus reached 100%, the virus was harvested. The purified target virus was screened by plague with different concentrations of ganciclovir （GCV） and the recombinant virus was identified by plague reduction test and DNA sequencing of drug-resistant genes （UL97 and UL54）. Results The UL97 fragments containing intended mutations for transfection were constructed successfully. After eotransfected with AD169 DNA mixture for 7 days, rHCMV formed cytopathology was obviously visible, which was verified as rHCMV infected focus by HCMV-PP65 antigen test. The UL97 genotypic analysis of recombinant virus obtained by cloning was as expected. No mutation was found by UL54 gene sequencing. The GCV susceptibility of rHCMV positive clone was 15 μmol/L （50 % inhibiting concentration）, which was 12-fold of standard AD169 strain and was drug-resistant phenotype. Conclusions The rHCMV containing intended mutations is constructed successfully by cotransfection into MRC-5 cells and the recombinant virus strain is obtained by GCV screening and plaque purifying. The establishment of this method provides technique platform for identifications of new drug-resistant mutations of HCMV during anti-viral therapy.